ES2343082T3 - A METHOD FOR PROVIDING AND ANALYZING AN ANIMAL POPULATION THAT HAS AN ESSENTIALLY IDENTIAL METABOLOMA. - Google Patents

Abstract

The present invention relates to a method for providing an animal population having an essentially identical metabolome comprising compiling an animal population being of essentially the same age, keeping said animal population for a time period sufficient for acclimatization under the following housing conditions: (i) constant temperature, (ii) constant humidity, (iii) physical separation of the animals of the animal population, (iv) feeding ad libitum, wherein the food to be fed is essentially free of chemical or microbial contaminance, (v) drinking libitum, wherein the drinking liquid is essentially free of chemical or microbial contaminance, (vi) constant illumination period, and providing the animal population after said time period. Further, the present invention relates to methods for the identification of a compound which effects the metabolome of an animal, or methods for the identification of a marker for such compounds. Moreover, the present invention encompasses methods for the identification of such compounds or markers thereof comprising metabolically analyzing a sample from at least one animal of an animal population.

Description

A method to provide and analyze a population animal that has an essentially identical metabolome.

The present invention relates to a method for providing a population of mammalian animals that have an essentially identical metabolome comprising the compilation of a population of mammalian animals being essentially the same age, as said population of mammalian animals has for a period of time. sufficient for acclimatization under the following housing conditions: (i) constant temperature, (ii) constant humidity, (iii) physical separation of mammalian animals from the mammalian animal population, (iv) ad libitum feeding, where the food to be offered it is essentially free of chemicals or microbial contaminants, (v) ad libitum drinking liquid, where the drinking liquid is essentially free of microbial chemicals or contaminants, (vi) constant period of illumination, and provide the mammalian animal population after said time frame. Additionally, the present invention relates to methods for the identification of a compound which has an effect on the metabolome of a mammalian animal, or methods for the identification of an indicator for such compounds. In addition, the present invention encompasses methods for the identification of such compounds or indicators thereof comprising metabolically analyzing a sample of at least one mammalian animal from a mammalian animal population.

State of the art phenotype analysis techniques organisms include, inter alia, complete genome analysis of said organism, called genomic, analysis of all proteins, called proteomic, and the analysis of all transcripts of RNA More recently, these fundamental analysis techniques Phenotypic have been completed by metabolome analysis, all metabolites, of an organism. This analysis is called metabolomic or, sometimes, metabonomic. It can be defined metabolomics as the qualitative and quantitative determination of all compounds of low molecular weight (i.e. metabolites) in the cells of an organism or body fluid at a time specific and under specific environmental conditions. An advantage of metabolomics is that effects caused by exogenous factors can be monitored immediately due to metabolic changes that usually appear much earlier than changes, if any, in the proteome or even in the genome.

Several techniques have already been described for Metabolome analysis of an organism. These techniques include, for example, mass spectroscopy, NMR, infrared spectroscopy with Fourier transforms (FT-IR), and detection by flame ionization (FID), optionally paired with techniques by chromatographic separation such as liquid chromatography, gas chromatography or HPLC. These techniques allow exploration of high yield of large populations of organisms for variations in the composition of your metabolome, that is, these allow to determine a metabolic phenotype. The metabolic phenotype of an organism are all its metabolites (metabolome) in a certain way moment and is the result of complex interactions of your genetic composition and the environment in which the organism lives. Correspondingly, differences in the metabolome of individuals from a population of organisms can be caused not only by differences in the genome but also by environmental factors that They influence metabolic activities. The metabolomics, therefore, allows to determine even effects of exogenous factors that do not they immediately influence the genome, transcription or proteome of a organism. For example, a toxic compound is harmful to a organism but should not necessarily cause changes in the genome of said organism. Currently, laboratory animals of a mammalian animal population are kept individually low equal and controlled environmental and nutritional conditions (US-A-5163380, US-A-346488).

At present, a disadvantage in the metabolomic compared to other approvals of principle of phenotypic analysis is that organisms and, in particular, animals mammals that are used in metabolic studies in the art previous do not share a common metabolome at the beginning of the study. In genomic, for example, a population that has a genome essentially identical can be easily provided by tip cloning techniques. However, it would be highly desirable to detect metabolic influences of exogenous factors, such as toxic compounds or drugs under conditions optimized Techniques for establishing mammalian animals that have an essentially identical metabolome are the basis for an analysis of Reliable and efficient metabolome. Notwithstanding the need for consequently, such techniques are not yet described.

Therefore, the underlying technical problem to the present invention should be seen as the provision of methods to meet the aforementioned needs, ie provide an animal mammalian population which has a metabolome essentially identical suitable for reliable studies of Metabolome and reliable analysis. The technical problem is solved. by the modalities characterized in the claims and described below.

Accordingly, the present invention is relates to a method to provide an animal population of mammals  which have an essentially identical metabolome comprising:

to)

Compile an animal population of mammals being essentially the same age;

b)

Maintain said animal population of mammals of step a) for a sufficient first period of time for acclimatization under the following conditions of accommodation:

i)

Constant temperature;

ii)

Constant humidity;

iii)

Physical separation of animals mammals of the mammalian animal population;

iv)

Feed ad libitum , where the food to be offered is essentially free of chemical or microbial contaminants;

v)

Drink ad libitum , where the drinking liquid is essentially free of chemical or microbial contaminants;

saw)

Constant lighting period; Y

C)

Provide the animal population mammal from step b) after said first period of weather.

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The term "method of providing" as used hereafter, preferably, does not encompass methods of treating the body of the mammalian animal. Specifically, the method referred to herein is not appropriate for medical treatment or therapy of any disease or disorder nor is it appropriate to improve or maintain the general welfare of mammalian animals of the mammalian animal population compared to mammalian animals kept under other physiological conditions In addition, the term does not include any reproduction technique per se .

The term "mammalian animal population" relates to the plurality of mammalian animals. A plurality of mammalian animals as used herein is a group of mammalian animals consisting of at least, preferably, 5 to 120, more preferably 5 to 25 of said mammalian animals by sex, dose and point in time. Mammalian animals of the mammalian animal population are of the same species and, preferably, of the same strain. Preferred mammalian animals for use in the method of the present invention are rodents, more preferably, rats or mice. If rats are used for the method of the present invention, it is further preferred that these rats be wistar rats (CrlGlxBrlHan: Wi) (Charles RIver, USA). Additional preferred rat strains are: BDIX strains; BDIX / CrCrl, BDIX / OrlCrl, Brown Norway strains; BN / CrlCrlj, BN / Crl, BN / OrlCrl, BN / OrlCrl, BN / SsNHsdMcwiCrl, Buffalo strain; BUF / CrCrl; Fischer strains, F344 / DuCrl, F344 / DuCrlCrlj, F344 / lcoCrl, F344 / DuCrl, SASCO Fischer strain, F344 / NCrl; strains Copenhagen, COP / CrCrl, COP / NCrl; Cotton strains, COT / NCrl strain; Dahl / SS, SS / JrHsdMcwiCrl; Fawn Hooded strain, FHH / EurMcwiCrl; strain GK, GK / Crlj; Lewis strains, LEW / CrlCrl; LEW / Crl; Noble strain, NBL / CrlCrl; strain SHR, SHR / NCrlCrlj, SHR / NCrl, SHR / NCrl; strain WAG, WAG / RijCrl; strains Wistar Furth, WF / CrCrl, WF / lcoCrl; strains WKY, WKY / NCrl, WKY / NCrlCrlj, WKY / NlcoCrl; strains ZDF, ZDF / Crl-Leprfa, ZDF / Crl-Leprfa; strains CD, Crl: CD (SD), Crlj: CD (SD), Crl: CD (SD), Crl: CD (SD); strain SASCO SD, Crl: SD; OFA strains, Crl: OFA (SD), Crl: OFA (SD) -hr; strain DIO, Crl: CD (SD) DR; strain DR, Crl: CD (SD) DR; strain Donryu, Crlj: DON; strain LEC, Crlj: LEC; Strains Wistar, Crlj: Wl, Crl: Wl; Strains Wistar Han, Crl: Wl (Han), Crl: Wl (Han), Crl: Wl (Han), Crl: Wl (Han); strain Wistar WU, Crl: Wl (WU) or any strain that is derived from the above-mentioned strains by means of cross-breeding or genetic manipulation. The aforementioned strains are well known in the art and commercially available, for example, through Charles River, USA or Harlan, USA. If mice are used for the method of the present invention, it is preferred that the mice be C57BL / GNCrl mice (Charles River, USA). Other preferred mouse strains are: CD-1 strains, Crlj: CD1 (ICR), Crl: CD1 (ICR), Crl: CD1 (ICR), Crl: CD1 (ICR), Crl: CD1 (ICR); strains CF-1, Crl: CF1, Crl: OF1; CFW strains, Crl: CFW (SW); strains NMRI, Crl: NMRI, Crl: NMRI (Han); strain PGP, Crl: CF1-Abcb1a, strains SKH1, Crl: SKH1-hr, Crl: SKH1-hr; strain SKH2, Crl: SKH2-hr; strain A / J, A / J; strains 129, 129S2 / SvPasCrl, 129S2 / SvPasCrl; 129 / S1 / Sv-p + Tyr + Kitsl-J / Crl, 129 / s1 / svlmJ; AKR strain, AKR / NCrl; strains BALB / c, BALB / cAnNCrlCrlj, BALB / CAnNCrl, BALB / cByJ; strains C3H, C3H / HeJCrl, C3H / HeNCrlCrlj,
C3H / HeNcrl, C3H / HeOuJ; strains C57BU6, C57BU6JCrl, C57BL / 6J, C57BL / 6JCrl. C57BL / 6NCrlCrlj, C57BL / 6NCrl, C57BL / 6J, C57BU6JCrl, C57BL / 6J; strains C57BU10, C57BL / 10JCrl, C57BL / 10JCrl; CBA, CBA / CaCrl strains,
CBA / J, CBA / JNCrlj, CBA / JNCrljCrlg; strain CB17, CB17 / lcrCrl; strains DBA / 1, DBA / 1JCRL, DBA / 1JNCrlj,
DBA / 1NlcoCrl; strains DBA / 2, DBA / 2J, DBA / 2NCrlCrlj, DBA / 2NCrl, DBA / 2JCrl; strain FVB, FVB / NCrl; strain NC, NC / NgaTndCrlj; strain NOD, NOD / LtJCrl; strains SJL, SJL / JOrlCrl, SJL / JOrl / CrlCrlj, SJUJ, SJL / JCrl; strains B6C3F1, B6C3F1 / Crl, B6C3F1 / Crlj; B6CBAF1 strains, B6CBAF1 / Crl, B6CBAF1 / J, B6CBAF1 / Crl; BDF1 (B6D2F1) strains, B6D2F1 / Crl, B6D2F1 / J, B6D2F1 / Crlj; strain B6SJLF1, B6SJLF1 / J; strain C3D2F1, C3D2F1 / Crl; CDF1 strains
(CD2F1), CD2F1 / Crl, CD2F1 / Crlj, CD2F1 / Crl; strain CBAB6F1, CBAB6F1 / Crl; strain CB6F1, CB6F1 / Crl; strain
NMRCF1, NMRCF1 / Crl or any strain that is derived from the aforementioned strains through cross-breeding or genetic manipulation. The aforementioned strains are well known in the art and commercially available, for example, through Charles River, USA or Harlan, USA. Additional preferred mammalian animals are dogs. Preferred dogs include beagles, more preferably, Beagle strains HsdRdg: DOBE or HsdHFr: DOBE or strains that are derived from the above-mentioned strains by means of cross-breeding or genetic manipulation. The aforementioned strains can be purchased from Harlan, USA. Other preferred beagles are from an inbred strain and can be purchased from BASF AG, Germany. However, the mammalian animals to be used are not limited to the aforementioned mammalian animals and can be subsequently selected from a group consisting of: cats, horses, cows, sheep, goats, rabbits.

The term "metabolome" as used here refers to all metabolites in a cell, tissue, organ or The whole mammalian animal. The metabolites are preferably small molecule compounds, such as enzyme substrates of metabolic routes, intermediates of such routes or products obtained by a metabolic route. Metabolic pathways are good. known in the art or may vary between species. Preferably, said pathways include at least the cycle of citric acid, respiratory chain, photosynthesis, photo respiration, glycolysis, gluconeogenesis, hexose route monophosphate, oxidative pentose phosphate pathway, production and β-oxidation of fatty acids, cycle of urea, amino acid biosynthesis pathways, degradation pathways of protein such as proteasomal degradation, degradation pathways of amino acids, biosynthesis or degradation of: lipids, polyketides (including for example flavonoids and isoflavonoids), isoprenoids (including for example terpenes, sterols, steroids, carotenoids, xanthophylls), carbohydrates, phenylpropanoids and derivatives, alkaloids, benzenoids, indoles, compounds of indole-sulfur, porphyrins, anthocyanins, hormones, vitamins, co-factors such as groups prosthetics or electron vehicles, lignin, glucosinolates, purines, pyrimidines, nucleosides, nucleotides and molecules related such as tRNAs, microRNAs (miRNAs) or mRNAs. By consequently, metabolites of small molecule compounds are preferably compounds of the following classes of compounds: alcohols, alkanes, alkenes, alkynes, aromatic compounds, ketones, aldehydes, carboxylic acids, esters, amines, imines, amides, cyanides, amino acids, peptides, thiols, thioesters, phosphate esters, sulfate esters, thioethers, sulfoxides, ethers, or combinations or derivatives of the compounds above mentioned. Small molecules between metabolites can be primary metabolites which are necessary for everything normal functioning, organ function or animal growth, development or health In addition, small molecule metabolites subsequently they comprise secondary metabolites that have function ecological essential, for example metabolites that allow a organism adapt to its environment. In addition, metabolites are not limited to said primary and secondary metabolites and additionally encompass artificial compounds of small molecules, such artificial compounds of small molecules are derivatives of small molecules exogenously provided which are administered or assimilated by an organism but are not primary or secondary metabolites as defined above. By example, artificial compounds of small molecules can be metabolic products obtained from drugs by metabolic pathways of mammalian animal. In addition, metabolites additionally include peptides, oligopeptides, polypeptides, oligonucleotides and polynucleotides, such as RNA or DNA. More preferably, a metabolite has a molecular weight of 50 Da (Dalton) to 30,000 Da, more preferably less than 30,000 Da, less than 20,000 Da, less that 15,000 Da, less than 10,000 Da, less than 8,000 Da, less than 7,000 Da, less than 6,000 Da, less than 5,000 Da, less than 4,000 Gives, less than 3,000 Da, less than 2,000 Da, less than 1,000 Da, less than 500 Da, less than 300 Da, less than 200 Da, less than 100 Gives. Preferably, a metabolite has, however, a weight molecular of at least 50 Da. More preferably, a metabolite according to the present invention it has a molecular weight of 50 Gives up to 1,500 Da.

The term "metabolome essentially identical "means that all individuals in the population mammalian animal provided by the method of the present invention have synchronized metabolic activities resulting (i) in the presence of essentially the same metabolites in the metabolome of each individual in the population and (ii) in amounts of said metabolites which are essentially identical for each of individuals of the mammalian animal population. Preferably, synchronized metabolic activities as used here means that all metabolic pathways that affect the metabolome of mammalian animals are active in essentially the same cells, tissues or organs at essentially the same time, levels of genetic expression in all mammalian animals are essentially identical and artificial small molecules are available to all mammalian animals in essentially identical quantities. It is to be understood that the amounts of metabolite may vary among individuals in the animal population Mammalian within certain limits. If the same metabolites are present in essentially the same amounts in individuals of the mammalian animal population referred to in according to the present invention, it can be determined by several analysis techniques of qualitative and / or quantitative compounds. These techniques include but are not limited to techniques. chromatographic for separation of compounds together with techniques of analysis of compounds such as mass spectrometry (MS), Ion resonance spectroscopy with Fourier transforms (FT-IR), or FiD. In accordance with the present invention a quantitative and / or qualitative determination of the metabolites using gas and liquid chromatography along with mass spectrometry (LC-MS and GC-MS). Details of such preferred methods are described below. Preferably, an animal population of mammals it has an essentially identical metabolome if mass spectra generated by one of these techniques are essentially identical for each individual in the population. These mass spectra are essentially identical if all the important peaks that are detectable by, for example, a commercial annotation algorithm pico, such as ChemStation (Agilent Technologies, USA), Analyst (MDS SCIEX, Canada) or AMDIS (NIST, USA), appear in all spectra at essentially identical retention times chromatographic As discussed above, minor variations are tolerable if they do not result in statistical differences significant. If a variation is minor according to the present invention can be determined by statistical algorithms  appropriate well known to the person skilled in the art such as principal component analysis (PCA) or minimum tests partial squares (PLS). Preferably, a metabolome essentially identical of the mammalian animals of the population Mammalian animal can be determined along with an analysis of multiple variables (for example PCA) or hierarchical grouping.

The term "compilation" as used herein refers to selecting mammalian animals from any source to establish the mammalian animal population to be subjected to the method of the present invention. Therefore, mammalian animals may be progeny of the same mammalian animal mother or progeny of different mammalian animal mothers. In the event that a single progeny of an animal mammalian mother is used as a source, the entire progeny can be used for the compilation of the animal mammalian population or mammalian animals selected from the progeny can be used. Compiling as used here is performed with respect to the age of mammalian animals, that is, all individuals in the population must be essentially the same age as described below in detail. However, additional features can be taken into consideration. Additionally, such as weight, sex, appearance in general (ie only healthy animals per appearance can be selected
swims)

The term "essentially the same age" means that mammalian animals have a comparable state of development, for example mammalian animals can be embryos, Youth or adults. A preferred age of mammalian animals at to be used in the method of the present invention is an age of teenage stage, preferably the young adolescence stage. Mammalian animals of the mammalian animal population, preferably, they have an age with the range of X ± n day, where X is the expected age of the mammalian animal population and n is selected from an integer from 1 to 5 days, and, more preferably, n is 1 day. In other words, a mammalian animal given population must be at most for a day greater or less that the average age of mammalian animals in the population mammalian animal More preferable, all mammalian animals of the population are of age X. Such mammalian animals can be provided by compiling mammalian animals that are progeny of a litter, that is, littermates, or are compiled from Different litters of the same day. In case they are going to be used embryos, it is to be understood that essentially the same age is It relates to its stages of development. The stages of development of Embryos of various species can be determined by techniques well known in the art. They can be calculated, for example, based on the point in fertilization time. In addition, in case that embryos are used, it should be additionally understood that pregnant mothers who have such embryos should be kept in conditions referred to here.

If rats or mice are used like animals mammals in the method of the present invention, it is preferred that mammalian animals are of age X ± 1 day, where X is selected from an integer from 10 to 100 days, plus preferably, an integer from 20 to 80 days, and, more Preferably, X is 63, 64 or 65 days after birth. Plus Preferably, X is 64 days after birth. If dogs are used as mammalian animals in the method of the present invention, X is preferably 6 months after birth.

The term "keep" as used by according to the method of the present invention it refers to the particular conditions of accommodation, food, drink and environmental factors that are applied to mammalian animals of the mammalian animal population. It is preferred that mammalian animals are kept under conditions as set forth in the Guide for The OECD Chemical Test No: 407. In addition, particular conditions They are described as follows.

i)

Everybody the mammalian animals of the mammalian animal population are kept under the same constant temperature. Care must be taken to choose a temperature to perform the method of this invention that does not cause stress to mammalian animals. Preferably, the temperature should be between 20 to 24 ± 2 ° C, more preferably 22 ± 2 ° C, more preferably 22, 23 or 24ºC.

ii)

In addition, all mammalian animals of the mammalian animal population are kept under the same humidity constant. The humidity should be at least 30%, but it should not exceed 70%. However, in rare rare situations (such as during room or cage cleaning) moisture can exceed 70%. Preferably, the humidity is 50-60%

iii)

Be has found that the physical separation of mammalian animals from the mammalian animal population is also important for the method of the present invention. Therefore, each mammalian animal of the mammalian animal population must be kept in a space separated, for example, a separate cage.

iv)

Mammalian animals of the mammalian animal population are fed ad libitum . The food to be used must be essentially free of chemical or microbial contaminants. The standards to be applied are set forth in Fed. Reg. Vol. 44, No. 91, May 09, 1979, p. 27354. More preferably, microbial contaminants such as bacteria are below 5 x 10 5 cells per g of food. Such food can be purchased from Provimi Kliba SA Kaiseraugst (Switzerland) as ground food Kliba maintenance diet for mouse / rat "LPG".

v)

Mammalian animals of the mammalian animal population are supplied ad libitum with a liquid to drink. Preferably, said liquid is water. However, other water-based liquids can also be used. Such liquids may comprise, for example, nutrients, vitamins or minerals that are required by animals. If water is used as the drinking liquid, the water must be free of chemical and microbial contaminants as set forth in the European Drinking Water Directive 98/83 / EG.

saw)

Finally, each mammalian animal of the Mammalian animal population must be subjected to the same periods lighting constants Constant illumination is achieved, preferably, by artificial lighting (solar spectrum normal). The lighting period is 12 hours of light followed by 12 hours of darkness. After the lighting period begins again. A preferred lighting period, therefore, is 12 hours of light, from 6:00 to 18:00, and 12 hours of darkness, from 6:00 p.m. to 6:00 a.m.

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Accommodation conditions previously mentioned can be applied to mammalian animals using a common storage space for cages comprising physically separated mammalian animals. Said common space of storage can be a room or animal house mammals Manque has all the mammalian animals of the population in the same room, can be easily achieved constant humidity, temperature and lighting period regulating These parameters for the whole room or house. The regulation of these parameters is preferably assisted by automation and The parameters are constantly monitored.

Under the term "first period of time enough for acclimatization "it must be understood that animals  mammals of the mammalian animal population must be kept under the particular housing conditions previously mentioned for a period of time that allows synchronization of the metabolic activities of mammalian animals so that mammalian animals are acclimatized and have essentially the same metabolome. Specifically, said first period should be the long enough to allow all individuals of the population adopt the same circadian rhythm, rhythm of food digestion, or periods of stillness / movement. In addition, the first period of time should allow each mammal animal adjust your biochemical and physiological parameters in response to applied environmental conditions, such as humidity and temperature. Preferably, said first period of time has a duration of 5 to 10 days, more preferably 6 to 8, and more preferably 7 days.

In the studies underlying this invention, it has been surprisingly found that a population mammalian animal that has essentially the same metabolome can be provided by the method of the present invention. The superior results achieved by such method depend, without However, in strictly obeying the housing conditions previously mentioned and the criteria for compiling the mammalian animal population, ie compile with respect to age of the mammalian animals of the animal population. The last issue It is particularly surprising because according to the OECD Guide No: 407, loc. cit., care must be taken regarding weight or sex when mammalian animals are selected for purposes Analytical Advantageously, using the method of the present invention, it is possible to generate a mammalian animal population that It can be applied for comparative metabolomics. Because of essentially identical metabolome it is possible to study reliably and efficiently, for example, toxic effects of compounds or determine forms of action of compounds such as drugs or drug candidates. In addition, the method of the present invention can be easily implemented in facilities since existing mammalian animals and is therefore profitable.

The present invention also encompasses a method. for the identification of a compound that has an effect on the metabolome of a mammalian animal comprising:

to)

Provide an animal population mammal using the steps of the method of claim one;

b)

Manage said animal population mammal a compound that is suspected to have an effect on the metabolome of a mammalian animal; Y

C)

Analyze the population's metabolome mammalian animal from step b);

d)

Perform a necropsy of each of Mammalian animals of the mammalian animal population.

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The term "identification" as used here means that the method of the present invention must be applied to identify or explore compounds that may have effect on the metabolome of a mammalian animal. Therefore, it is expected that the method will provide data that allows identification of a compound that has an effect on the metabolome of a mammalian animal. Such data can be obtained by several techniques to analyze the metabolome of a described mammalian animal in detail below. The data can be in the form of raw data or Processed data. Preferably, the result of the analysis of the metabolome in the form of raw data or processed data will be compared to corresponding data obtained from a reference. Therefore, it is to be understood that identification as used according to the method of the present invention may require additional steps However, these additional steps are related to well known techniques in comparative analysis including those mentioned before and below. Further, identification as used herein preferably encompasses identification of the property of the compounds to cause a metabolic effect of a mammalian animal, in principle, despite the type of effect or mode of action. In addition, the term covers additionally, preferably, the identification of the type of effect that is provoked or even the precise mode of action. For the therefore, the method of the present invention can also be used to identify a particular metabolic pathway influenced by a compound or certain mode of action of a compound. Plus preferably, the method for identification described can be used above and below.

The term "compound that has an effect on the metabolome of a mammalian animal "encompasses all kinds of chemical compounds. Preferably, a compound as used here is a compound of small molecules, a peptide, a polypeptide or a polynucleotide. Small molecules as is done reference according to the present invention include molecules inorganic and organic having a low molecular weight, preferably a molecular weight that is lower than 30,000 kDa, more preferable lower than 20,000 Da, 10,000 Da, 8,000 Da, 5,000 Da, 3,000 Da, 2,000 Da, 1,000 Da or 500 Da. More preferable, it suspects that said compound is a toxic compound, a nutrient, a nutraceutical or therapeutically active compound (i.e. a drug). A drug according to the present invention will have a therapeutic effect on the mammalian animal, that is, it will treat or It will improve a medical condition. Said medical condition may be a disease or condition or poor welfare of an animal mammal. Improvement or treatment can be monitored by the appearance or degree of the symptoms of said disease, disorder or deficiency. Drugs according to the present invention also include drug precursors that are converted in vivo to drugs therapeutically active and compounds suspected of being drugs, is Say drug candidates. Peptides and polypeptides to be used of according to the method of the present invention include peptides and naturally occurring polypeptides as well as peptides and artificial polypeptides Peptides and polypeptides that occur naturally they can be obtained from all kinds of organisms including plants, animals, fungi, bacteria or viruses. Peptides and artificial polypeptides can be generated, for example, with random peptide synthesis techniques. Peptides or Preferred polypeptides to be used in accordance with the present invention are those that directly or indirectly influence metabolic activities or metabolic pathways in a mammalian animal of according to the present invention. More preferably, said polypeptides or peptides have a therapeutic value, such as peptide hormones, growth factors, survival, cytokines, receptors for said polypeptides, biologically active antibodies or fragments thereof. Polynucleotides to be used according to the method of Present invention are RNA or DNA molecules. Preferably, said polynucleotides encode peptides or polypeptides that directly or indirectly influence metabolic activities or routes metabolic For example, appropriate polynucleotides can encode enzymes, preferably those of metabolic pathways, peptides or polypeptides as specified above or peptides or polypeptides that regulate the expression of the peptides or aforementioned polypeptides, such as factors of transcription. In addition, polynucleotides to be used in the method of the present invention as compounds may be polynucleotides that interfere with gene expression (i.e. transcription or translation), such as anti-sense RNA molecules, anti-sense oligonucleotides or small RNAs from interference (siRNA) to be used for interference technology RNA (RNAi).

The compounds referred to in according to the present invention they are explored for their ability to have effects on the metabolome of a mammalian animal. For the therefore, a compound within the meaning of the present invention will alter the metabolome composition of a mammalian animal as soon as administer Said effect on the metabolome of a mammalian animal It can be a qualitative or quantitative effect. An effect qualitative about the metabolome as used here means that by at least one metabolite of the mammalian animal's metabolome is absent or at least one additional metabolite is present after administration of the compound. A quantitative effect as used in accordance with the present invention means that the amount of at least one metabolite is altered, that is more high or lower, after administration of said compound. It is to be understood that a compound that has an effect on the The metabolome of a mammalian animal can also influence the biological function of the cells, tissues or organs of said  mammalian animal and may cause poisoning, improvements in health, deterioration of health or conditions.

The term "administer" as used here includes all the techniques by which the compounds can be systemically provided to the mammalian animal. In addition, the term covers techniques for administering compounds to suspected sites of potential action such as a target tissue or organ, that is topical administration The compounds to be administered from according to the present invention may be comprised in a subsequent composition comprising appropriate vehicles, such as pharmaceutical vehicles, excipients and / or diluents. Examples for well known diluents include saline solutions buffered with phosphate, water, emulsions, such as emulsions oil / water, various types of wetting agents, solutions sterile, etc. Administration of the compounds or of the appropriate compositions mentioned above may be performed in different ways, for example by administration intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial. Preferably, the administration is achieved by oral administration, but preferably the compound is mixed with the drinking liquid or food. Appropriate treatment and dosage regimens are well known to the person skilled in the art and a regime of Preferred treatment and dose is described in the Examples. Preferably, the mammalian animals of the animal population test mammals (ie the population of mammalian animals to which must be administered the compound) are subdivided into at least one group of males and at least one group of females and at least one high dose group and at least one group of low dose

The term "analyze the metabolome" as used in accordance with the present invention refers to techniques to quantitatively or qualitatively analyze the metabolome. In a first step, said qualitative or quantitative analysis of metabolome comprises determining qualitatively or quantitatively the metabolome composition, that is the metabolites. Means and methods to qualitatively analyze the metabolome comprise those who are able to determine the presence or absence of the metabolites comprised by the metabolome. Means and methods to quantitatively analyze the metabolome are those that determine the amount of said metabolites. It is to be understood that there are means and methods that can determine the presence or absence as well as the amount of metabolites and, so both, allow a combination of qualitative analysis or quantitative. In addition, in accordance with the methods herein invention is not necessarily required to determine all metabolome metabolites Rather, metabolome analysis can be performed by determining the presence or absence or the amount of a portion of the metabolites found to be characteristic, a predetermined series of metabolites or a metabolic profile for the metabolome Characteristic or predetermined metabolites they comprise known metabolites as well as known ones unknown The latter are metabolites that are barely known by its signal in, for example, mass spectra. Nature chemistry of such unknown acquaintances, however, is not Precisely known. A metabolic profile as used here is relates to all kinds of unique identifier for a certain metabolome, that is a fingerprint of a metabolome. Analysis Qualitative or quantitative metabolome is preferably performed using compound analysis techniques. Dispositives appropriate for such determination of compounds are well known in the technique Preferably, mass spectrometry is used in particular gas chromatography mass spectrometry (GC-MS), liquid chromatography spectrometry of mass (LC-MS), infusion mass spectrometry Direct or mass spectrometry with Fourier transforms ion-cyclotron-resonance (FT-ICR-MS), capillary electrophoresis mass spectrometry (CE-MS), spectrometry of mass together with high performance liquid chromatography (HPLC-MS), quadrupole mass spectrometry, any sequentially matched mass spectrometry, such as MS-MS or MS-MS-MS, mass spectrometry of inductively paired plasma (ICP-MS), pyrolysis mass spectrometry (Py-MS), ion mobility mass spectrometry or mass spectrometry of flight time (TOF). More preferable, LC-MS and / or GC-MS are used as described in detail below. Such techniques are disclosed in, for example, Nissen, Journal of Chromatography A, 703, 1995: 37-57, US 4,540,884 or US 5,397,894. As an alternative or in addition to techniques of mass spectrometry the following techniques can be used for compound determination: nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), infrared analysis with Fourier transforms (FT-IR), spectroscopy ultra violet (UV), refractive index (IR), detection by fluorescence, radiochemical detection, electrochemical detection, light scattering (LS), Raman dispersive spectroscopy or detection by flame ionization (FID). These techniques are well known. for the person skilled in the art and can be applied without further Preamble

In an additional step, analyze the metabolome as used herein, preferably includes a comparison of metabolites, amounts of metabolites or metabolic profile at a corresponding reference. This reference can be derived from a metabolome analysis of a mammalian animal population to which you have not been given any compound suspected of causing effects on the metabolome of a mammalian animal, through which is preferred that this mammalian animal population of reference has been otherwise treated identically. Later appropriate references for a comparison according to the The present invention can be derived from an analysis of metabolome of mammalian animals to which they were administered known compounds have an effect on the metabolome. Such compounds can be compounds that are toxic or that are therapeutically active through a known mode of action. From this way, using mammalian animals treated with the compounds mentioned above have an effect on the metabolome as a reference, it can not only be determined if A new compound has an effect on the metabolome in general. Rather, it can be determined later if said new compound causes toxic effects or can be therapeutically active through a certain mode of action or has at least the potential to do it. Preferably, the data resulting from a Metabolome analysis of the references described above are saved and provided in the form of an appropriate database. In addition, data from a metabolome analysis performed by the method of the present invention for new compounds may also serve as a reference for other new compounds and may be also saved and provided by said database for later analysis. Compare as used here encompasses comparison of raw data obtained by metabolome analysis or any type of processed data derived from said raw data. Means and Suitable methods for comparing data are well known in the technique and include, for example, component analysis principal (PCA) or partial least squares tests (PLS). In principle, any statistical test that allows to determine if metabolites, amounts thereof or a metabolic profile as is described above differ significantly between different mammalian animals or time points of determination is appropriate to make the comparison referred to here. The differences mentioned above may, in principle, be determined by model recognition algorithms, algorithms of statistical test and / or multivariable algorithms, for example, Principal Component Analysis (PCA), Component Analysis Simple (SCA), Independent Component Analysis (ICA), Regression Main Component (PCR), Partial Minimum Squares (PLS), Discriminant Analysis PLS (PLS-DA), Machines Support Vector (SVM), Neural Channels, Bayesian Channels, Bayesian Learning Channels, Mutual Information, Channels Backpropagation, Symmetric Direct Feed Channels, Maps of Self-Organization (SOMs), Genetic Algorithms, Hierarchical or K-Media Grouping, Anova, T-Test of Students, Kruskal-Wallis Test, Test Mann-Whitney, Test Tukey-Kramer or Try the Best of Hsu. Data processing as it is done reference above may preferably include a step of data validation. In said data validation step, data Inconsistent are excluded from further analysis. Data inconsistent can result in technical problems during qualitative or quantitative determination of the composition of the metabolome Therefore, it is expected that the technical parameters of the devices used for determination are monitored constantly and provided for data validation in a appropriate database. Subsequent data validation can be achieved with internal validation tools that evaluate statistically each measured value or parameter of a series of data. For example, if mass spectrometry is used to analyze the metabolome, a series of raw data that comprises peaks is generated. These peaks appear at a certain position in a space three-dimensional consisting of a time range retention, intensity range and index range mass-to-load (m / z). Each one of Such peaks can be evaluated and confirmed by algorithms of peak validation such as ChemStation (Agilent Technologies, USA), Analyst (MDS SCIEX, Canada) or AMDIS (NIST, USA).

In addition, data related to mammalian animals or accommodation can be taken into consideration for validation of data. For example, if accommodation conditions vary, the metabolome of a mammalian animal of the mammalian animal population It can be adversely influenced. Such metabolome adversely influenced by a mammalian animal can, however, cause false positive or negative results. Therefore it is also preferred to monitor data related to mammalian animals or to accommodation as described below in detail and take into account this data during data validation.

In addition, data processing, preferably, includes standardization of raw data with respect to internal references For example, peaks that can be assigned to Known metabolites can be used to normalize spikes of unknown metabolites within a series of data obtained to a metabolome of an individual from the mammalian animal population with with respect to, for example, signal strength.

In addition, data processing can be subsequently required to create consistent data by converting data in a numerical format, converting the data to a unit of common format and / or reducing data dimensionally. The data can be subsequently integrated into that information relating them to the sample or mammalian animals that is assigned to this. Appropriate means and methods to create such consistent data are disclosed in WO 03/046798.

The data processing steps and the comparison referred to above are preferably assisted by automation, for example, by a program of proper computer that runs on a computer. Computer it is preferably operatively connected to several bases of data referred to in accordance with this invention. Therefore, in one embodiment, the present invention It is also related to a database or database system of data comprising the results obtained by the method of present invention More preferable, such a database or system of databases includes information related to Metabolomas obtained by compounds suspected to affect the metabolism of an organism, such as toxic compounds or therapeutically active (ie drugs). Thus, the basis of data can be used as a tool (database of reference) for exploration tests which are aimed to identify new drugs or toxic compounds by their way of action reflected by the metabolome.

It is to be understood that the analysis of metabolome must be performed after a period of time enough to allow the compound to enter its cells effectors, tissues or organs, that is a period of time of incubation. In other words, the analysis must be performed. after the compound has become bioavailable. According to the chemical nature of the compound, the right person in the technique will know what period of time should be sufficient for the bioavailability of said compound. Such a period of time can Also be defined in a pilot experiment. For example, a compound being of similar or identical chemical nature can be connected to a detectable label. Said labeled compound It can then be administered to a mammalian animal. Time until the label is detectable in effector cells suspects, tissues or organs will be determined and will serve as incubation time period for the method herein invention.

The metabolome analysis too, preferably, it comprises taking a sample of each mammalian animal of the mammalian animal population that will be subsequently analyzed as described above. Suitable samples include cells, tissues or animal organs or body fluids including blood, plasma, serum, urine, spinal liquor. It is well known in the art, how Such samples can be taken. Appropriate techniques include blood sampling, biopsy, liquor sampling, classification cell phone, etc. A preferred sampling program is described in the examples. The sample can be subjected to pre treatments. Such pre treatments include enzymatic digestion of material biological that is not appropriate for metabolome analysis, extraction procedures to obtain certain metabolites of the sample, fractionation of the sample, for example, to be able to obtain polar and / or non-polar metabolite fractions, or formation of metabolite derivatives, for example, prior to chromatography Such pretreatment techniques are well known. in the technique and can be applied by the right person in the technique without further ado.

Advantageously, due to providing the Mammalian animal population according to the present invention is You can perform reliable comparative metabolome analysis. The results of said reliable analysis according to the method of the present invention will make drug discovery faster and toxicological assessments Preferably, databases are created that comprise metabolic data (ie data on the qualitative and quantitative composition of metabolomes or their profiles metabolic) for a mode of action of a compound or data metabolics that reflect the administration of toxic compounds or drugs The metabolic data of said databases can be used as references when using the method herein invention for an exploration of subsequent compounds that are Suspicion have an effect on the metabolome. It is planned that each scanning will produce a more comprehensive view on the modes of action. In addition, by comparison with said metabolic data (data reference) toxic compounds or therapeutically compounds assets can be quickly identified. Preferably, the method of the present invention is performed in a high format performance.

In a preferred embodiment of the method of present invention, the mammalian animal population is maintained by a second period of time after administering to the population mammalian animal compounds suspected of having an effect on the metabolome in step b) under housing conditions described to which reference is made above.

More preferable, the analysis of step c) is carried out at least once during said second period of time or at least three times during the second period of time, where the second and any additional analysis is performed after a period of time that is twice the period of time that passed since the previous analysis. Plus Preferably, the first analysis is performed seven days after the administration of the compound in step b).

It has been found in the study underlying the present invention that it is advantageous to evaluate the metabolome on a time range from acute effects to long term effects including intermediate time points. Therefore, the method of the present invention allows repeated measurements during a investigation. Therefore, the analysis of the animals Mammals is preferably performed at least once, preferably twice or more preferably at least three times during the second period of time. The time points for analyzes have to be selected to include early effects acute as well as chronic or long-term effects. Points of appropriate time for analysis can be determined by the right-handed person in art depending on the mammalian animal that is analyzed without further ado. Specifically, if an animal rodent mammal is used, such as a population of rats or mice, a time course of analysis is preferred in which the second and Any additional analysis is performed 7 days after the administration of the compound, the second analysis should be performed after 14 days and the third analysis must be performed  after 28 days after administration. Preferably, if the analysis is performed analyzing a fluid sample body, such as blood, it is preferred that mammalian animals of the mammalian animal population will be maintained for a period of fasting (i.e. food withdrawal) for approximately 16 to 20 hours before blood sampling. Also, perform the analysis during a constant time window it allows to compensate for metabolic changes that can be caused for example by rhythm circadian of the mammalian animals of the animal population Mammal Therefore, it is preferred that blood sampling and analysis be performed within and at a constant time window For all the analyzes. Preferably analysis including Samples are taken between 7.30 and 10.30 a.m. In addition, the methods of the present invention may additionally encompass in addition to the subsequent blood metabolome analysis investigations of metabolome of different organs or body fluids of Mammalian animals of the mammalian animal population.

In addition, in addition to the investigations of metabolome, the method may comprise additional research that are not related to the metabolome. Specifically, it is expected that each mammalian animal of the mammalian animal population will be pathologically investigated after the metabolic analysis is completed Such research includes animal necropsy. including careful examination of the external surface of the body, all holes, cranial, thoracic and abdominal and its contents. In addition, a pathological analysis of liver, kidneys, adrenal, testicles, epididymis, thymus, spleen, brain and heart should be performed as is appropriate and as described in Guidelines No. 407, loc. Cit., for example. In addition, histopathological examination should be preferably performed as described in the following:

The following tissues should be preserved in the most appropriate fixation medium for the type of fabric and for the intended examination subsequent histopathology: all gross lesions, brain (representative regions including brain, cerebellum and bridge brainstem), spinal cord, stomach, small intestines and thick (including Peyer's plaques), liver, kidneys, adrenal, spleen, heart, thymus, thyroid, trachea and lungs (preserved inflating with fixative and then immersion), gonads, sexual organs accessories (eg uterus, prostate), urinary bladder, nodules lymphatics (preferably a lymph node that covers the path of administration and other distant from the administration path for cover systemic effects), peripheral nerves (sciatic or tibial) preferably close to the muscle, and a medulla section spinal (or, alternatively, a spinal cord aspirate fresh prepared). Clinical and other discoveries discoveries may suggest the need to examine tissues additional. Also any body considered likely to be target organ based on the known properties of the compound a proof must be preserved.

In addition, it is also preferred that parameters hematological and biochemical of mammalian animals of the Mammalian animal population be determined. It is preferred that data related to pathology and hematological parameters as well as biochemists are stored in a database appropriate for each of the mammalian animals in the population mammalian animal as data related to mammalian animals. The Stored data is preferably used for the evaluation of metabolic data generated by metabolome analysis and / or for data processing. In a preferred mode of the method of the present invention, taking into account these data related to mammalian animals allows to avoid false results positive or false negative because mammalian animals that must compared to its parallels in a mammalian animal population a abnormal pathology, hematology or abnormal biochemical parameters may be excluded from the data evaluation during the data processing. In addition, it is subsequently preferred to include data related to mammalian animals on body weight, consumption of food, drinking fluid and clinical signs such as also possible abnormalities that may appear when mammalian animals are maintained for the first and second period of time as specified above.

Thus, in another preferred mode of method of the present invention, said method comprising additionally monitoring of body weight, food consumption, drinking fluid consumption and clinical signs of the population mammalian animal Such monitoring may, more preferably, be assisted. by automation and monitored body weight data, food consumption, drinking fluid consumption and clinical signs they are collected in a database for each of the animals mammals of the mammalian animal population.

In addition, in view of the above, the method of present invention preferably consists of monitoring of abnormalities for each mammalian animal of the animal population Mammal The term "abnormalities" as used herein is refers to abnormalities that can be easily detected by maintenance staff, ie abnormalities that usually do not They require monitoring by a doctor. More preferable, the abnormalities  are automatically monitored and the data obtained from said monitoring are stored in a database for each individual of The mammalian animal population.

As referenced above, in a preferred embodiment of the method of the present invention, said analysis includes comparing the metabolome of the animal population Mammal with a reference.

More preferable, comparing comprises generating a metabolic profile of the mammalian animal metabolome of the mammalian animal population and comparing said profile with a reference. More preferable, a difference in profiles Metabolic is indicative for a compound that has an effect on The metabolome of a mammalian animal. It is to be understood that the reference in this most preferred modality is a metabolic profile derived from an untreated mammalian animal.

The term "metabolic profile" means that a specific fingerprint is established during analysis for each metabolome of a mammalian animal of the animal population Mammal Said metabolic profile can be derived from minus one signal (for example a peak in the mass spectrum) obtained from a mammalian animal or a sample thereof by the method of the present invention. More preferable, a profile Metabolic is derived from a plurality of said signals. The signals can be obtained from a single metabolite or from a plurality of metabolites. It is to be understood that the signals primary can be further processed by techniques appropriate as described above. Preferably, a series of three-dimensional data is generated using at less a time-resolved separation technique and at least a technique of resolved separation. Such a series of data could be obtained, for example from chromatography together with spectrometry of mass as described above. Such a series of data three-dimensional can be analyzed by algorithms conventional peak determination, such as ChemStation or AMDIS, which allow the specific detection of maximum and / or minimum in said three-dimensional data series. The maximum and minimum thus extracted will be the metabolic profile specific for the metabolome of a certain mammalian animal of a mammalian animal population. In addition, the extracted signals, such as the maximum or minimum of the peaks can be later processed in characteristic values as a function of respective time and mass. It is to be understood that the profiles metabolic generated by the aforementioned techniques preferably consist of data series that are dimensionally reduced and therefore can easily be compared to each one for statistical tests, including principal component analysis (PCA) or minimum tests partial squares (PLS). A difference in metabolic profile of a metabolome of a test mammalian animal (ie a mammalian animal that has obtained a compound suspected of having effect on the metabolome of a mammalian animal) and a reference it is an indicator that the compound effectively induces changes metabolic in the test mammalian animal. However as already described above, this does not necessarily mean that the composition  quantitative metabolome (ie the number of compounds that are present) will be changed. Changes in metabolic profiles it can also be an indicator for a quantitative composition impaired metabolome (i.e. the amount of compounds that are present is altered). More preferable, the profiles Metabolic can be compared to metabolic profiles comprised of a database with reference profiles for known drugs, pro-drugs or toxic compounds or Its modes of action. Advantageously, the method of the present invention therefore allows procedures drug exploratives and drug candidates are more efficient in time since toxic or harmful effects of compounds can be determined at an early stage of Drug development Subsequently, toxicity tests may be easily carried out efficiently in time since the Start of toxic events can be monitored by changes in metabolic profiles In particular, this is advantageous for compounds that cause long-term toxic effects instead of immediate effects

It is to be understood that differences or changes in The metabolome of a mammalian animal can also be analyzed comparing specific metabolites of known chemical nature or unknown instead of a metabolic profile.

Therefore, in another preferred embodiment of the method of the present invention, compare comprises comparing at least one metabolite metabolite of an animal population Mammal with a reference. More preferable, a difference in said at least one metabolite is indicative for a compound that It has an effect on the metabolome of a mammalian animal. Is to be understood that the reference in this most preferred modality is a metabolic profile derived from an untreated mammalian animal.

In another preferred embodiment of the method of present invention and in light of the foregoing, said compound is a compound suspected of being toxic or a compound suspected of being toxic A drug.

The present invention additionally relates to a method for identifying a marker for a compound that has an effect on the metabolome of an animal mammal, comprising the steps of the method of the present invention as described above and the additional step of providing a marker for said compound based on metabolome analysis. In a preferred embodiment of the method of the present invention, said marker is a metabolic profile of the animal population mammal that is altered compared to a reference. Plus Preferably, said marker indicates toxicity of a compound, a mode of action of a compound or a therapeutic activity of a compound.

In addition, the present invention encompasses a method to identify a compound that has an effect on the metabolome of a mammalian animal that comprises metabolic analysis of a sample of at least one mammalian animal from an animal population mammal to which a compound suspected of having effect on the metabolome and where said mammalian animal population has been maintained before and after the administration of composed under the housing conditions to which it was made reference above.

The term "metabolically analyzing" as it is used here, it preferably covers all means, methods and modalities described above to analyze the metabolome of a mammalian animal.

The term "sample" as used here covers samples of biological material obtained from the mammalian animal To be investigated. Appropriate sources for samples have already been described above.

In a preferred embodiment of the method of present invention, said analyzes comprise comparing the metabolome of the sample of a mammalian animal of said population Animal with a reference.

More preferable, comparing comprises generating a metabolic profile for the sample of a mammalian animal of the mammalian animal population and comparing said profile with a reference. More preferable, a difference in metabolic profile It is indicative for a compound that has an effect on the metabolome of a mammalian animal. It is to be understood that the reference in this preferred embodiment is a metabolic profile derived from a untreated mammalian animal.

Also more preferable, compare comprises compare at least one metabolite metabolite of a sample of a mammalian animal of the mammalian animal population with a reference. More preferable, a difference in the one mentioned by less a metabolite is indicative for a compound that has an effect on the metabolome of a mammalian animal. It is to be understood that the reference in this most preferred mode is a profile metabolic derived from an untreated mammalian animal.

Also encompassed by the present invention is a method for identifying a marker for a compound that has an effect on the metabolome of a mammalian animal that comprises the steps of the above mentioned methods (based on the samples) and the additional step of providing said marker based on the metabolome analysis

In a preferred embodiment of said method of present invention, said marker is a metabolic profile or is by at least one metabolite of an animal's sample metabolome mammal of the mammalian animal population that is altered compared to a reference. More preferably, said marker indicates toxicity of a compound, a mode of action of a compound or an activity Therapeutic of a compound.

The invention will now be illustrated by the Following Examples. However, the Examples are simply for purpose of illustration and it is not the purpose to limit the field of application of the invention

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Example 1 Animal Maintenance

Rats of the strain CrlGlxBrlHan: Wi were purchased from Charles River, Sulzfeld, Germany who is aged from 63 to 65 days. Consecutively, each animal has been marked with A tattoo on the ear. The animals were kept under following housing conditions:

Air conditions:

Temperature 20-24ºC, humidity 30-70%. Any deviation has been documented.

Lighting Period:

12 hours of light from 6.00 to 18.00 hours, 12 hours of darkness from 6:00 p.m. to 6:00 a.m.

Type of cage:

Wire cages, type DK III, BECKER & Co., Castrop-Rauxel, Germany.

No. of animals per cage:

one

Type of diet:

Ground maintenance diet Kliba for mouse / rat "LPG", flour, supplied by Provimi Kliba SA, Kaiseraugst (Switzerland), ad libitum

Water:

Drink water ad libitum

Acclimatization:

During the 7-day acclimatization period, the animals have been accustomed to the environmental conditions of Study and diet.

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Example 2 Metabolic investigation of test compounds

Female and male wistar rats have been randomized and assigned to dose groups before the onset of administration period based on their weights. The animals have been treated with five different test compounds at a level of high dose and low dose according to the following program shown in Table 1

TABLE 1

one

Blood sampling was performed as is. indicated in the following time program shown in the Table 2.

TABLE 2

2

During the experiment, an animal review dying and dead has been performed twice, daily from Monday to Friday and once a day on Saturday, Sunday and holidays. The Animals will be checked daily for any clinical sign abnormal. Abnormalities and changes will be documented for each animal.

Food consumption has been determined in the Study days 6, 13, 20 and 27. Drinking water consumption has been Revised daily within the general observations. The weight body has been determined before the start of the period of administration, to be able to randomize the animals. During the administration period body weight has been determined in the days of the study 0, 6, 13, 20 and 27. The average daily consumption of the test substances has been calculated based on the values individual for body weight and food consumption.

Means and standard deviations have been calculated using the Dunnet test.

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Blood sampling was performed as follow:

Before necropsy or blood sampling, it withdrew the food for approximately 16 to 20 hours (period of fast). Blood sampling was performed between 7:30 and 10:30 A.M. The blood was taken from the venous plexus retro-orbital of anesthetized animals with isoflurane From each animal 1 ml of blood will be collected in tubes EDTA plastics as anticoagulant (10 µl of a solution to 10%) Blood samples are centrifuged. The plasma of each Sample is separated and transferred to another plastic tube. The precipitated erythrocytes are washed three times with 0.9% NaCl, and filled to 1 ml with sterile distilled water (Ampuwa®, Fresenius, Bad Homburg, Germany) to hemolyze red blood cells. Hemoglobin is determined in hemolyzed blood samples (40 µl hemolyzer plus 160 µL of 1.5% NaCl) with an analyzer automatic (ADVIA 120, Bayer AG, Fernwald, Germany). Blood, plasma and hemolysed are sampled and stored in Eppendorf tubes originals Transport and sample preparation is performed under ice cooling. At the end of the preparation of the sample all samples are superimposed with an atmosphere of pure nitrogen, sealed with "Parafilm M" and stored at -80 ° C (under nitrogen atmosphere) until further processing of samples (for example, shipping).

After completing the experiment, it determined the clinical pathology for each animal. To this end all  the animals that survived the study have been sacrificed for decapitation under anesthesia by isoflorano (if provided final blood sampling) or by anesthesia with CO2.

Claims (30)

1. A method to provide an animal population mammal that has an essentially identical metabolome that understands:

to)

compile a mammalian animal population being essentially the same age;

b)

maintain said animal population mammal from step a) for a first period sufficient to acclimatization under the following conditions of accommodation:

i)

constant temperature;

ii)

constant humidity;

iii)

physical separation of animals mammals of the mammalian animal population;

iv)

feed ad libitum , where the food to be provided is essentially free of chemical and microbial contaminants;

v)

drink ad libitum , where the drinking liquid is essentially free of chemical or microbial contaminants;

saw)

constant lighting period; Y

C)

provide the mammalian animal population from step b) after said first period of time.

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2. The method of claim 1, wherein said Mammalian animal population is a population of rodents. 3. The method of claim 2, wherein said Rodent is a rat or mouse. 4. The method of claim 3, wherein said age is within the range of X ± 1 day, where X is the age visualized of the population. 5. A method for identifying a compound that has an effect on the metabolome of a mammalian animal  which includes:

to)

provide a mammalian animal population using the steps of the method of claim 1;

b)

administer to said animal population mammal a compound suspected of having an effect on the metabolome  of a mammalian animal;

C)

analyze the population metabolome mammalian animal from step b); Y

d)

perform the necropsy of each of Mammalian animals of the mammalian animal population.

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6. The method of claim 5, wherein the Mammalian animal population is maintained for a second period of time after administering to the mammalian animal population a compound suspected of being toxic in step b) under the housing conditions described in claim 1. 7. The method of claim 6, wherein the analysis of step c) is performed at least once during said second period of time. 8. The method of claim 6, wherein the analysis of step c) is performed at least three times during the said second period of time where the second and subsequent analyzes are performed after a period of time which is twice the period of time since the previous analysis. 9. The method of claim 8, wherein the First analysis is performed seven days after administration  of the compound in step b). 10. The method of any of the claims 5 to 9, further comprising monitoring of the body weight, food consumption, drinking fluid consumption and clinical signs of the mammalian animal population. 11. The method of any of the claims 5 to 10, further comprising monitoring of abnormalities for each mammalian animal of the animal population Mammal 12. The method of any of the claims 5 to 11, wherein the analysis comprises comparing the metabolome of the mammalian animal population with a reference. 13. The method of claim 12, wherein the comparison comprises generating a metabolic profile for the metabolome of a mammalian animal of a mammalian animal population and comparing said profile with a reference. 14. The method of claim 13, wherein a difference in metabolic profiles is indicative for a compound that has an effect on the metabolome of an animal mammal. 15. The method of claim 12, wherein compare comprises the comparison of at least one metabolite of the metabolome of a mammalian animal of the mammalian animal population with a reference. 16. The method of claim 15, wherein a difference in the saying at least one metabolite is indicative for a compound that has an effect on the metabolome of a mammal. 17. The method of any of the claims 5 to 16, wherein said compound is a compound suspected of being toxic or is a compound suspected of being a drug. 18. A method for identifying a marker for a compound that has an effect on the metabolome of a mammal comprising the steps of the method of any of the claims 5 to 17 and the additional step of providing a marker for said compound based on metabolome analysis. 19. The method of claim 18, wherein said marker is a metabolic profile or is at least one metabolite of a mammal of the mammalian animal population that is altered compared to a reference. 20. The method of claim 18 or 19, wherein said marker indicates the toxicity of a compound, a mode of action of a compound or a therapeutic activity of a compound. 21. A method to identify a compound that has an effect on the metabolome of a mammal that comprises metabolically analyze a sample of at least one mammal from a mammalian animal population to which a compound suspected of having an effect on the metabolome of a mammal and where said mammalian animal population has been maintained before and after administration of the compound under the housing conditions described in claim 1. 22. The method of claim 21, wherein said analysis comprises comparing the metabolome of the sample of a mammal of said mammalian animal population with a reference. 23. The method of claim 22, wherein compare comprises generating a metabolic profile for the sample of an animal of the mammalian animal population and comparing said profile With a reference. 24. The method of claim 23, wherein a difference in metabolic profile is indicative for a compound It has an effect on the metabolome of a mammal. 25. The method of claim 22, wherein compare comprises comparing at least one metabolite of sample metabolome of an animal population with a reference. 26. The method of claim 25, wherein a difference in the saying at least one metabolite is indicative for a compound that has an effect on the metabolome of a animal. 27. The method of any of the claims 21 to 26, wherein said compound is a compound Toxic or drug 28. A method for identifying a marker for a compound that has an effect on the metabolome of a mammal comprising the steps of the method of any of the claims 21 to 26 and the additional step of providing said marker based on metabolome analysis. 29. The method of claim 28, wherein said marker is a metabolic profile or is at least one metabolite metabolite of the sample of a mammal of the mammalian animal population that is altered compared to a reference. 30. The method of claim 28 or 29, when said marker indicating toxicity of a compound, a mode of action of a compound or a therapeutic activity of a compound.